Polypeptide and anti-HIV agent prepared therefrom

ABSTRACT

A polypeptide represented by formula (I), and one example of such polypeptide be represented as formula 1 is presented. The above presented polypeptide may be useful in a pharmaceutical composition as an antimicrobial or antiviral agent, specifically as an anti-HIV agent and as a component of the DNA-transfecting systems for gene therapy.

1. FIELD OF THE INVENTION

This invention relates to a novel polypeptide(s) or a pharmaceuticallyacceptable salt thereof exhibiting a strong affinity tolipopolysaccharides, particularly endotoxins. The polypeptide may beused in a pharmaceutical composition as an anti-viral agent (e.g.anti-HIV agent).

2. BACKGROUND OF THE INVENTION

Two families of antimicrobial polypeptides have been isolated fromhorseshoe crabs which exhibit an affinity to endotoxins (see, forexample, Shigenaga et al., 1990, J. Biol. Chem. 265:21350-21354; Kawanoet al., 1990, J. Biol. Chem. 265:15365-15367; Muta et al., 1990, J.Biochem. 108:261-266; Japanese Laid-Open Patent Publication No.167230/1990; Japanese Laid-Open Patent Publication No. 152987/1990;Japanese Laid-Open Patent Publication No. 53799/1990; Published SearchedApplication 500194/1990; Miyata et al., 1989, J. Biochem. 106:663-668;Akaji et al., 1989, Chem. Pharm. Bull. 37:2661-2664; Tokunaga andIwanaga, 1989, Taisha(Metabolism) 26:429-439 ; Shieh et al., 1989, FEBSLett. 252:121-124; and Nakamura et al., 1988, J. Biol. Chem.263:16709-16713).

One family, the tachyplesin family has been isolated from the Japanesehorseshoe crab Tachypleus. Three tachyplesins, I, II, and III have beenidentified. A second family, the polyphemusin family has been isolatedfrom the American horseshoe crab, Limulus polyphemus. Two polyphemusins,I and II have been identified; their amino acid sequences are shown inFIG. 1.

The polypeptides in both families consist of 17 or 18 amino acidresidues and have four conserved regions in common and two disulfidebridges (see FIG. 1).

Both tachyplesins and polyphemusins have been found to inhibit thegrowth of both Gram-negative and -positive bacteria at lowconcentrations as well as fungi, such as Candida albicans and formcomplexes with bacterial lipopolysaccharides (Shigenaga et al., 1990, J.Biol. Chem. 265:21350-21354 and Muta et al., 1990, J. Biochem.108:261-266). Also, the polypeptides in a tachyplesin family have beenfound to exhibit some inhibition activity for virus, such as Influenzavirus, vesicular stomatitis virus (Murakami et al., 1991, Chemotherapy,37, 327-334) or human immunodeficiency virus (Morimoto, et al., 1991,Chemotherapy, 37, 206-211). It is very interesting that such apolypeptide with above properties may be one of the key substances whichenables the horseshoe crab to adapt with changes in their externalenvironment and to preserve their species from ancient times to now as aliving fossil.

On the other hand, with respect to the survival of the highly evolvedhuman beings, development of such drugs is extremely longing that have aprophylactic or therapeutic effect on acquired immune deficiencysyndrome (AIDS) caused by infection with human immunodeficiency virus(HIV).

Present inventors and coresearchers have been studied a correlationbetween molecular structure and anti-HIV activity on the polypeptideswith changing or modifying the amino acid of components, taking noticeof these polypeptides which are related to the long preservation of thehorseshoe crabs. As a result, the past new polypeptides had beeninvented by us, fundamentally different from the common structure ofsuch the known polypeptides of the horseshoe crabs.

Surprisingly, it was found that these past new polypeptides have anexcellent bioactivity whose anti-HIV values are at least 5 times or morehigher than that of a known polypeptides of the horseshoe crabs.

Following references had be seen, Nakashima et al., 1992, Antimicrob.Agents Chemother., 36, 1249-1255 ; Masuda et al., 1992, Biochem.Biophys. Res. Commun., 189, 845-850 ; Tamamura et al., 1993, Chem.Pharm. Bull., 41, 978-980 ; Tamamura et al., 1993, Biochim. Biophys.Acta, 1163, 209-216 ; Masuda et al., 1992, J. Pharmacobio. Dyn., 15,s-90 ; International Laid-Open Publication WO 92/04374 ; JapaneseLaid-Open Patent Publication No.163298/1993.

(Hereinafter, among the new polypeptides, the compound T-22 is referredto the best mode compound of the representatives.) Having examined thestructural requirements for exhibiting the activity of the newpolypeptide consisting of 16 to 18 amino acids as represented by theT-22 compound, the inventors and coresearchers arrived at some inventiveconcepts of a minimum essential structure.

In general, when an exogenous peptide of relatively high molecularweight is administered into human body, it is often recognized as analien substance by the self defence function of the human body. As aresult, it is likely to become an antigenic substance. When used for amedical purpose, it is desirable that a peptide-based bioactivesubstance may be a compound of low molecular weight in order to reduceits likelihood of being recognized as an alien substance. It is alsorequired that the substance may have a high potency.

The T-22 compound was found to be a polypeptide consisting of 18 aminoacid residues. The objective of our investigation was to maintain thesame level of anti-HIV potency as that of the T-22 compound, whilereducing the number of amino acid residues. As a result, we succeeded inreducing the number of residues by 4 (four). As long as the compound hasthe basic structure, its activity does not decline even when a specificregion is modified. Moreover, by the said modification, we discoveredthe novel polypeptide having such essential structure that can provide abroader range of physicochemical characteristics, and also a broaderselection in therapeutic methods, i.e. increasing/decreasing of thehydrophilicity and lipophilicity (affinity to lipid); selectiveaccumulation in a specific organ and/or cell; and increasing/decreasingits residence time within the human body; and possible development offormulations.

3. SUMMARY OF THE INVENTION

The present invention relates to a novel polypeptide(s) which is derivedfrom the past new polypeptides with a high anti-HIV activity inventedbased on the known high endotoxin affinity polypeptides of horseshoecrabs, but has a significant difference.

The past new polypeptides consist of 16-18 amino acid residues, 4cysteine or 2 systine residues and antiparallel β sheet structure with βturn as a turning position. Like the past new polypeptides, thepolypeptides of the present invention have an antiparallel β sheetstructure with possibly β turn located in the X at 7th position.However, in the polypeptides of the present invention, the numbers ofamino acid residues and cystein residues are reduced by four and two,respectively. Moreover, biological activity does not decrease even whena specific region is modified. The polypeptides of the present inventionmay be used as an anti-HIV reagent and as a component of theDNA-transfecting systems for gene therapy. As will be detailed in theSection 6, infra, the polypeptides of the invention have anti-HIV valuesthat are significantly higher than the past new polypeptides derivedfrom the known high endotoxin affinity polypeptides of horseshoe crabs.

3.1 DEFINITIONS

Peptide sequences defined herein are represented by three letterabbreviations for amino acid residues and substituted amino acidresidues as follows:

Ala (alanine); Arg (arginine); Cys (cysteine); Ile (isoleucine); Gly(glycine); Leu (leucine); Lys (lysine); Orn (ornithine); Phe(phenylalanine); Pro (proline); Trp (tryptophan); Tyr (tyrosine); Val(valine); DArg (D-arginine); DLys (D-lysine); DOrn (D-ornithine); Ac-Arg(N-α-acetyl arginine); FTC-Arg (N-α-fluororescein thiocarbamoylarginine); Laur-Arg (N-α-lauroyl arginine); Myr-Arg (N-α-myristoylarginine); Nicot-Arg (N-α-nicotinoyl arginine); Oct-Arg (N-α-octanoylarginine); Parm-Arg ((N-α-palmitoyl arginine); Parm-Orn (N-α-palmitoylornithine); PTC-Arg (N-α-phenyl thiocarbamoyl arginine); ε-N-Ac-DLys(ε-N-ω(ε-N-ωaminoacetyl-D-lysine) and ε-N-But-DLys(ε-N-ω-aminobutyryl-D-lysine).

The following terms, as used herein, will have the meanings indicated:

HIV=human immunodeficiency virus (all variants)

MOI=multiplicity of infection

SI=selectivity index (ratio of CC₅₀ to EC₅₀)

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequences of Tachyplesin I, Tachyplesin II,Tachyplesin III, Polyphemusin I, and Polyphemusin II. Conserved aminoacids are boxed. The disulfide linkages between Cys-3 or -4 and Cys-16or -17 and Cys-7 or -8 and Cys-12 or -13 are shown by solid lines.

FIG. 2 shows a synthetic scheme for synthesizing polypeptide (1) of theinvention.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is accomplished based on the above aspects, and isrelated to a novel polypeptide represented by the following formula

    A.sub.1 -Trp-Cys-A.sub.2 -A.sub.3 -A.sub.3 -X-A.sub.2 -A.sub.3 -A.sub.3 -Cys-A.sub.3 -A.sub.4                                     (I)

or salt thereof in which

A₁ is a basic amino acid residue, or a peptide residue having one or atleast two basic amino acids, selected from the group consisting oflysine, arginine and ornithine, said basic amino acid residue or peptideresidue in which N-α hydrogen atom of amino terminal end of said aminoacid residue may be replaced with an acyl group or a substitutedthiocarbamoyl group, forming N-α acyl substituted basic amino acidresidue, N-α acyl substituted peptide residue, N-α substitutedthiocarbamoyl group substituted basic amino acid residue or N-αsubstituted thiocarbamoyl group substituted peptide residue;

A₂ is a tyrosine or phenylalanine residue;

A₃ is a lysine or arginine residue;

A₄ is an --OH (derived from a carboxyl group) or an --NH₂ (derived froman acid amide group);

X is a peptide residue selected from the group consisting of thepeptides represented by D-ornithyl-proline, prolyl-D-ornithine,D-lysyl-proline, prolyl-D-lysine, D-arginyl-proline, prolyl-D-arginine,glycyl-ornithine, ornithyl-glycine, glycyl-lysine, lysyl-glycine,glycyl-arginine and arginyl-glycine, in which the hydrogen atom of theω-amino group of D-lysine, L-lysine, D-ornithine and L-ornithine may bereplaced by an ω-amino acyl group, and said peptide residue is connectedto the amino acid residues at the 6th and the 8th positions via peptidebond per se;

Trp is a tryptophan residue; and

Cys is a cysteine residue; and the cysteine residues at the 3- and 11-positions may be linked through a disulfide linkage.

Specific examples of the polypeptides of the invention represented bythe formula (I), are shown in Table 1 and are numbered (1) to (25).

Each symbol denotes the corresponding amino acid residue by theinternationally admitted three-letter expression (see Section 3.0.,supra).

                                      TABLE 1                                     __________________________________________________________________________     ##STR2##                                                                      (2)Arg --Trp--Cys--Tyr--Arg--Lys --DLys--Pro --Tyr--Arg--Lys--Cys--Arg--N    H.sub.2                                                                        (3)Arg--Arg --Trp--Cys--Tyr--Arg--Lys --Lys--Gly --Tyr--Arg--Lys--Cys--Ar    g--NH.sub.2                                                                    (4)Arg --Trp--Cys--Tyr--Arg--Lys --Lys--Gly --Tyr--Arg--Lys--Cys--Arg--NH    .sub.2                                                                         (5)Arg--Arg --Trp--Cys--Tyr--Arg--Lys --Pro--DLys --Tyr--Arg--Lys--Cys--A    rg--NH.sub.2                                                                   (6)Arg--Arg --Trp--Cys--Tyr--Arg--Lys --DOrn--Pro --Tyr--Arg--Lys--Cys--A    rg--NH.sub.2                                                                   (7)Arg--Arg --Trp--Cys--Tyr--Arg--Lys --Pro--DArg --Tyr--Arg--Lys--Cys--A    rg--NH.sub.2                                                                   (8)Arg--Arg --Trp--Cys--Tyr--Arg--Lys --Gly--Lys --Tyr--Arg--Lys--Cys--Ar    g--NH.sub.2                                                                    (9)Arg--Arg --Trp--Cys--Tyr--Arg--Lys --Arg--Gly --Tyr--Arg--Lys--Cys--Ar    g--NH.sub.2                                                                   (10)Arg--Arg --Trp--Cys--Tyr--Arg--Lys --Gly--Arg --Tyr--Arg--Lys--Cys--Ar    g--NH.sub.2                                                                   (11)Lys--Arg --Trp--Cys--Tyr--Lys--Arg --DLys--Pro --Tyr--Lys--Arg--Cys--A    rg--NH.sub.2                                                                  (12)Lys--Lys --Trp--Cys--Phe--Arg--Lys --DLys--Pro --Phe--Arg--Lys--Cys--A    rg--NH.sub.2                                                                  (13)Arg--Lys --Trp--Cys--Tyr--Lys--Arg --DLys--Pro --Tyr--Arg--Arg--Cys--L    ys--NH.sub.2                                                                  (14)Ac--Arg--Arg --Trp--Cys--Tyr--Arg--Lys --DLys--Pro --Tyr--Arg--Lys--Cy    s--Arg--NH.sub.2                                                              (15)Oct--Arg--Arg --Trp--Cys--Tyr--Arg--Lys --DLys--Pro                       --Tyr--Arg--Lys--Cys--Arg--NH.sub.2                                           (16)Laur--Arg--Arg --Trp--Cys--Tyr--Arg--Lys --DLys--Pro                      --Tyr--Arg--Lys--Cys--Arg--NH.sub.2                                           (17)Myr--Arg--Arg --Trp--Cys--Tyr--Arg--Lys --DLys--Pro                       --Tyr--Arg--Lys--Cys--Arg--NH.sub.2                                           (18)Parm--Arg--Arg --Trp--Cys--Tyr--Arg--Lys --DLys--Pro                      --Tyr--Arg--Lys--Cys--Arg--NH.sub.2                                           (19)FTC--Arg--Arg --Trp--Cys--Tyr--Arg--Lys --DLys--Pro                       --Tyr--Arg--Lys--Cys--Arg--NH.sub.2                                           (20)PTC--Arg--Arg --Trp--Cys--Tyr--Arg--Lys --DLys--Pro                       --Tyr--Arg--Lys--Cys--Arg--NH.sub.2                                           (21)Nicot--Arg--Arg --Trp--Cys--Tyr--Arg--Lys --DLys--Pro                     --Tyr--Arg--Lys--Cys--Arg--NH.sub.2                                            ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                     __________________________________________________________________________

The above-mentioned polypeptide with a high anti-HIV activity consistsof 16-18 amino acid residues. The above-mentioned polypeptide with ahigh anti-HIV activity consists of 16-18 amino acid residues. In thebest mode compound (hereinafter abbreviated as "n-18 polypeptide" orT-22), it is considered that structural factors for exhibiting highactivity are essential of existence of 4(four) cysteine residues, offour or five aromatic amino acid residues, of eight basic amino acidresidues and of one glycine residue. Moreover, with respect to thepositional relationship of the n-18 polypeptide as show followingformula (A), the properties of the amino acid residues at 2- to 17-positions are tightly fixed. And at the first position of the n-18polypeptide, a structure-activity correlation was found in which therelative values of anti-HIV activity expressed increases with theincrease of the number of the amino acid residue.

The n-18 polypeptide is denoted as the following formula (A)

    A.sub.1 -A.sub.2 -Cys-A.sub.2 -A.sub.3 -A.sub.3 -Cys-A.sub.2 -A.sub.3 -Gly-A.sub.2 -Cys-A.sub.2 -A.sub.3 -A.sub.3 -Cys-A .sub.4 -A.sup.5(A)

in which

A₁ is no more than two amino acids selected from the group consisting oflysine and arginine;

A₂ is a tyrosine, phenylalanine or tryptophan residue;

A₃ is an arginine or lysine residue;

A₄ is at least one and no more than two amino acids selected from thegroup consisting of lysine and arginine;

A₅ is an --OH (derived from the carboxylic group); or an --NH₂ (derivedfrom the amide group);

Cys is a cysteine residue; and

Gly is a glycine residue.

In a specific embodiment, the cysteine residues at the 3- and 16-positions and/or the cysteine residues at the 7- and 12- positions maybe linked through a disulfide linkage (--S--S--).

In the n-18 polypeptide, the turning position with possibly beta-turnstructure is located at the 9- and 10-positions. And the peptide part ofthe 3-position to the 8-position and the peptide part of the 11-positionto the 16-position face each other.

Like the n-18 polypeptide, the polypeptides of the present inventionhave an antiparallel β sheet structure due to the existence ofintramolecular hydrogen bonding and disulfide linkage (--S--S--) withcysteine residues. While in the polypeptides of the present invention,the turning position with possibly β turn structure is so located in theX at 7th position that the peptide part of the 3-position to the6-position and the peptide part of the 8-position to the 11-positionface each other.

In the present invention, the relationship of the number of amino acidresidues at the 1- position of the formula of the present invention isthe same as that for the n-18 polypeptide.

It is confirmed that replacement of the hydrogen atom of an α-aminogroup of the N terminal amino acid residue at the said position by anacyl group or a substituted thiocarbamoyl group is important to exhibithigh anti-HIV activity of the novel polypeptide represented by thedenoted formula. By selecting different properties of the acyl or thesubstituted thiocarbamoyl group, it has become possible to give thenovel polypeptide of the invention hydrophilicity, affinity for lipids,distinct fluorescence properties, and etc. For example, the fluorescenceproperties of the fluorescein substituted thiocarbamoyl group in thepolypeptides of this invention can be used as a highly sensitivereporter dye for various purposes. See, for example, Brand and Witholtin "Methods in Enzymology," Vol. 11, page 776-856, ed. by Hirs, AcademicPress, New York, N.Y.(1967); Brand and Gohlke, 1972, Annu. Rev. Biochem.41:843-868; Stryer, 1978, Annu. Rev. Biochem. 47:819-846.

Moreover, it is very important and useful that the fluoresceinsubstituted thiocarbamoylated polypeptides of this invention couldexhibit even higher anti-HIV activity. Thus, the said polypeptides ofthis invention with fluorescence properties can be used as an importanttool for elucidating the manifestation-mechanism of anti-HIV activity ofthe polypeptides of this invention. For example, fate, metabolism ordistribution within body, organ, tissue or cell infected or non-infectedwith HIV after administration can be detected by fluorescencemicroscopy. In molecular level, information on the subtle conformationalchanges of the said polypeptides interacting with receptor moleculewithin the cells can be obtained by the use of the intrinsicfluorescence probes of the said polypeptides. Isolation andidentification of the receptor molecule itself may be possible by theuse of the fluorescence probes.

Furthermore, via an acyl group or a substituted thiocarbamoyl, it hasbecome possible to give the novel polypeptide bioactivity of compoundssuch as a sugar chain compounds, a lipid compounds, a nucleic acidcompounds, other kinds of peptides or proteins, etc. It plays animportant role in the activity manifestation of the novel polypeptide ofthe invention that the properties of the amino acid residue at the 4thto 6th positions and at the 8th to 10th positions of the formula of thepresent invention are the same repeated ones. With respect to formationof the three-dimensional structure of the novel polypeptide of thisinvention, an amino acid sequence order of the peptide is important thateasily makes peptide structural parts represented at 3rd to 7thpositions and 7th to 11th positions in the same planar structure inopposite manner and the peptide residue consisting of two amino acidsrepresented X at 7th position as a turning point. In case the cysteinesat position 3- and 7- are linked through a disulfide linkage, thefollowing three-dimensional structure formed by the peptide backbone ofthe novel polypeptide of this invention is important characteristics ofthe invention. That is to say; turn structure of the peptide residue Xat 7th position, which consists of a pair of glycine and basic aminoacid or of proline and D-form of basic amino acid (in principle,hydroxyproline may be substituted with proline having the same effect)is necessary factor to form the same planar structure with β- sheetstructure. The disulfide side chain linked 3- with 11- positions ofcysteine residues and the basic side chains of basic amino acid residuesat 5- and 9-positions are on the same side of backbone plane, while thearomatic side chains of aromatic amino acid residues at 4- and 8-positions and the basic side chains of basic amino acid residues at 6-and 10- positions are on the opposite side of backbone plane. Formationof these three-dimensional above mentioned structure is important. Thusthe novel polypeptide represented by above denoted formula was inventedwith such three-dimensional structure, resulting in reduction of 4(four)amino acid residues compared with n-18 polypeptide.

Furthermore, like the n-18 polypeptide(s), the polypeptides of thepresent invention exhibit very basic characteristics. Due to their basicnature, the polypeptide(s) of the present invention may form a salt byacid addition. For example, the polypeptide forms a salt with aninorganic acid (hydrochloric acid, hydrobromic acid, phosphoric acid,nitric acid, sulfuric acid or the like) or an organic carboxylic acid(acetic acid, halo acetic acid such as trifluoroacetic acid, propionicacid, maleic acid, succinic acid, malic acid, citric acid, tartaricacid, salicylic acid and an acidic sugar (glucuronic acid, galacturonicacid, gluconic acid, ascorbic acid or the like), an acidicpolysaccharide (hyaluronic acid, chondroitin sulfate, alginic acid orthe like) or an organic sulfonic acid (methanesulfonic acid,p-toluenesulfonic acid or the like) including sulfonic acid sugar estersuch as chondroitin sulfates.

The following is a more detailed description of the novel polypeptide ofthe present invention.

5.1 PREPARATION OF POLYPEPTIDES

The novel polypeptide of the invention can be prepared by methods knownin the art, for example, solid-phase synthesis techniques described in"Solid-Phase Peptide Synthesis", Stewart & Young, Pierce ChemicalCompany, Rockford, Illinois (1984). Namely, a straight-chain polypeptideof the invention having the above formula (I) can be obtained by linkingthe carboxyl group of an N-protected arginine at the 12-position to aninsoluble resin having amino groups directly attached or alternativelyattached through a spacer having a functional group capable of linkingto a carboxyl group (e.g. one capable of converting the carboxyl groupof arginine to a p-carboxymethylbenzylester). The amino group of theinsoluble resin having the arginine (Arg) residue at the 12-positionafter deblocking the N -protecting group is capable of successivelylinking, according to the solid phase method, with the respectiveprotected amino acids of the 11-position to the 1-position of the aminoacid sequence represented by the following formula (I) ##STR7## whereinA₁, A₂, A₃, A₄, Cys, Trp and X are as defined in the above formula (I)!.

In case that N-α acyl amino acid residue or N-α acyl peptide residue isselected in A₁ where the hydrogen atom at the N-α position of the aminoterminal amino acid residue is replaced by acyl groups, the N terminalamino group of the said peptide resin is acylated with the correspondingacid anhydride or the corresponding acid by use of condensing agents tothe acyl group to afford N-acyl peptide resin. Followed by cleavage ofthe insoluble resin and protecting groups on the amino acids, thestraight-chain polypeptide of the invention having the above formula (I)can be obtained. In case that N-α-substituted thiocarbamoyl amino acidresidue or N-α-substituted thiocarbamoyl peptide residue in A₁ of theaforementioned formula (I) where the hydrogen atom at the N-α positionof the amino terminal amino acid residue is replaced by substitutedthiocarbamoyl groups, the N-terminal N-α-substituted thiocarbamoylpolypeptide of the invention can be obtained by reaction with thesubstituted isothiocyanate compound under slightly alkaline conditions.

In this instance, the carboxyl terminus of the amino acid residue at the12-position can be either free (A₄ corresponds to --OH) or converted toan acid amide (A₄ corresponds to --NH₂). Further, in the obtainedpolypeptide, the two cysteines at the 3- and 11- positions can form adisulfide linkage (--S--S--) through the mercapto groups.

These disulfide linkage may be formed, for example, by air oxidation, orby the method of Atherton, E., et al., 1985, J. Chem. Soc., PerkinTrans. 1, 2065.

Unless otherwise indicated, the individual amino acid used in theaforementioned solid phase synthesis method is in the L-form, and thebasic amino acid coupled with a proline at the 7th position denoted by Xis limited to D-form.

Any insoluble resin having an amino group can be used in synthesizingthe novel polypeptide of the invention, as long as it can link throughits amino groups to the carboxyl group of the N-protected arginine orlysine at the C-terminus or in some cases to the carboxyl group of thespacer linked thereto and thereafter can be eliminated (removed).Examples of such insoluble resins include but are not limited toaminomethyl resins (aminomethylated styrene-divinylbenzene copolymers),benzhydrylamine resins, methylbenzhydrylamine resins andaminomethylphenoxymethyl resins and derivatives thereof. When abenzhydrylamine resin, methylbenzhydrylamine resin, dimethoxybenzhydrylamine (DMBHA) resin or aminomethylphenoxymethyl resin is used, an amideis directly obtained by cleavage, but an aminomethyl resin is preferredin view of yield.

As the spacer having a functional group capable of linking to a carboxylgroup or having a carboxyl group, there can, for example, be one that iscapable of converting the carboxyl group of arginine to ap-carboxymethylbenzyl ester, but there is no particular limitation onthe spacer.

The protected amino acid is an amino acid whose functional groups may beprotected with a protecting group using procedures known in the art, orvarious protected amino acids may be purchased commercially. Thoseskilled in the art will recognize that polypeptide synthetic methodsrequire the use of a protecting group to stabilize a labile side chainof an amino acid to prevent the side chain from being chemically alteredduring the synthesis process.

A protecting group for the a amino group of an amino acid is selectedfrom the group including but not limited to Boc (t-butyloxycarbonyl) orFmoc (9-fluorenylmethyloxy-carbonyl). A protecting group for theguanidino group of arginine (Arg) is selected from the group includingbut not limited to Tos (tosyl), NO₂ (nitro), Mtr (4-methoxy-2,3,6-trimethylbenzene sulfonyl) or Pmc(2,2,5,7,8-pentamethyl-chroman-6-sulfonyl). A protecting group for themercapto group of cysteine (Cys) may be selected from the groupincluding but not limited to Bzl (benzyl), MBzl (4-methoxybenzyl),4-MeBzl (4-methylbenzyl), Acm (acetamidomethyl), Trt (trityl), Npys(3-nitro-2-pyridinesulfenyl), t-Bu(t-butyl) or t-BuS (t-butylthio), andMbzl, 4-MeBzl, Trt, Acm and Npys are preferred. A protecting group forthe hydroxy group of tyrosine (Tyr) is selected from the group includingbut not limited to Bzl, C12Bzl (2,6-dichlorobenzyl) or t-Bu, and thehydroxy group may not be protected. A protecting group for the ε- aminogroup of lysine (Lys) is selected from the group including but notlimited to Z (benzyloxycarbonyl), ClZ (2-chloro-benzyloxycarbonyl), Bocor Npys. In case that the hydrogen atom of the side chain ε-amino groupof D or L lysine consisting the X at 7th position, the said amino groupmay be protected with aforementioned Z, ClZ, Boc or Npys. It ispreferred to select from these protecting groups an appropriate one thatis known to be suitable for the synthesis conditions.

The coupling of protected amino acids can be carried out according tocondensation methods known in the art, such as, for example, a DCC(dicyclohexylcarbodiimide) method, DIPCDI (diisopropylcarbodiimide)method Tartar, A. et al., 1979, J. Org. Chem. 44:5000!, active-estermethod, mixed or symmetrical acid anhydride method, carbonyldiimidazolemethod, DCC-HOBt (1-hydroxybenzotriazole) method Konig W. et al., 1970,Chem. Ber., 103:788, 2024, 2034! or diphenylphosphoryl azide method, butpreferably using the DCC method, DCC-HOBt method, DIPCDI-HOBt method orsymmetrical acid anhydride method. The condensation reaction may becarried out in an organic solvent such as dichloromethane,dimethylformamide, N-methylpyrrolidone (NMP) or a mixed solvent thereof.A deblocking reagent such as trifluoroacetic acid/dichloromethane,HCl/dioxane, piperidine/dimethylformamide (DMF) or NMP is used todeblock the protecting group for an a-amino group. The degree of theprogress of condensation reaction in each step of synthesis is pursuedby the method of E. Kaiser et al., 1970, Anal. Biochem., 34, 595 (theninhydrin reaction method).

According to the foregoing methods, a protected peptide resin having adesired amino acid sequence can be obtained. When an aminomethyl resinderivative is used as the insoluble resin, the protected polypeptide canbe removed from the resin, for example, by treating the protectedpeptide resin with ammonia in an appropriate solvent. The resultingprotected peptide is then treated with hydrogen fluoride to obtain apolypeptide amide represented by the above formula and freed of all theprotecting groups.

When a benzhydrylamine resin, methylbenzhydrylamine resin,aminomethylphenoxymethyl resin or DMBHA resin Funakoshi, S. et al.,1988, J. Chem. Soc., Chem. Commun., 382! is used as the insoluble resin,the polypeptide may be removed from the resin and the protecting groupscan simultaneously be removed from the polypeptide by treating theprotected peptide resin with hydrogen fluoride, TFMSA(trifluoromethanesulfonic acid) Yajima, H. et al.; "The Peptides" vol.5, page 65 (1983), published by Academic Press, edited by E. Gross;!,TMSOTf (trimethylsilyl triflate) Fujii, N. et al., 1987, J. Chem. Soc.,Chem. Commun., 274! or TMSBr (trimethylsilyl bromide) Fujii, N. etal.1987, Chem. Pharm. Bull., 35, 3880! or the like.

In a preferred embodiment, the resulting polypeptide is reduced with2-mercaptoethanol, DTT (dithio-threitol) or the like to make sure thatthe mercapto groups of the cysteines are in reduced form. The mercaptogroups may be subsequently oxidized to obtain a cyclic polypeptide.

The oxidation treatment can be carried out by a known method. Usually,such oxidizing agent as air or a ferricyanate (e.g. potassiumferricyanide) is used.

Alternatively, the polypeptide(s) of the invention may be produced usingrecombinant DNA technology. Accordingly, the nucleotide coding sequencesfor the polypeptide(s) of the invention may be cloned and expressedusing techniques well known in the art. See, for example, Maniatis etal., Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1991.

The polypeptides of the invention can be isolated and purified by meansknown in the art for polypeptides, for example, extraction,recrystallization, various chromatographies (gel filtration, ionexchange, partition, adsorption, reverse-phase), electrophoresis,counter-current distribution, etc., and reverse-phase high performanceliquid chromatography is the most effective.

5.2 USES FOR POLYPEPTIDES

The polypeptide(s) of the invention represented by the formula (I) havean ability to bind to endotoxins, an antibacterial activity, and anactivity to hemolyze endotoxin-sensitized hemocytes. Additionally, thepolypeptide(s) of the invention possess an antiviral activity. In aspecific embodiment, the polypeptide(s) of the invention have anti-HIVactivity. As will be detailed in Section 6, infra, the polypeptides ofthe invention exhibit significantly higher anti-HIV activity than knownhigh endotoxin affinity polypeptides (e.g., Tachyplesins I, II or III orPolyphemusins I or II) exhibits.

Recent development of delivery systems capable of efficientlyintroducing DNA into the target cell has made practical human genetherapy for genetic diseases, cancer, AIDS and etc. Morgan and Anderson,Annu. Rev. Biochem., 62, 191-217 (1993)!.

The DNA-transfection systems include the use of polycations, calciumphosphate, liposome fusion, retroviruses, microinjection,electroporation and protoplast fusion. However, all of these methodssuffer from one or more problems related to either cellular toxicity,poor reproducibility, inconvenience, or inefficiency of DNA deliveryFlegner et al., Proc. Natl. Acad. Sci. USA, 84, 7413-7417 (1987)!.

Recently, highly efficient DNA-transfection procedure using cationiclipid-DNA complex or cyclic amphipathic peptide-DNA complex Legendre andSzoka, Jr., Proc. Natl. Acad. Sci. USA, 90, 893-897 (1993)! has beenreported. The peptides that can form a transfecting complex with DNAinclude gramicidin S, tyrocidine, polymyxin B, polylysine and melittinall with cationic nature. Among these, the most effective cationicpeptide is gramicidin S which is known as an amphipathic cyclicdecapeptide antibiotic with β-sheet conformation and can permeabilizeand disrupt cell membranes. Both a positive charge and amphipathiccharacter of gramicidin S are thought to be important for the hightransfection. Considering these structural characteristics, thepolypeptides of this invention can be an alternative candidate ofgramicidin S for DNA complex with high transfecting ability because oftheir strongly cationic and amphipathic nature with β-sheetconformation.

In fact, tachyplesin I, one of the parent molecule of the polypeptidesof this invention, can permeabilize membranes and bind to DNA similarlyto gramicidin S Matsuzaki et al., Biochim. Biophys. Acta, 1070, 259-264(1991) and Yonezawa et al., Biochemistry, 31, 2998-3004 (1992)!.Moreover, the solution structure of T-22, one of the polypeptides ofthis invention (1), was confirmed to be very similar to that oftachyplesin I with amphipathic anti-parallel β-sheet structure Tamamuraet al., Biochim. Biophys. Acta, 1163, 209-216 (1993)!.

Therefore, the polypeptides of this invention may be used as a componentof the DNA-transfecting systems for gene therapy.

The polypeptide(s) of the present invention therefore may be used in apharmaceutical composition comprising the polypeptide(s) of theinvention or salt thereof and a pharmaceutically acceptable carrierselected in accordance with the administration method and administrationform of the pharmaceutical composition. The pharmaceutical carriers maybe such physiologically compatible buffers as Hank's or Ringer'ssolution, physiological saline, a mixture consisting of saline andglucose, and heparinized sodium-citrate-citric acid-dextrose solution.The pharmaceutical composition is orally or parenterally administered inaccordance with the object of treatment, and can be prepared as apreparation such as powder, granules, a solution for injection or oraladministration, tablets, suppositories, pessaries, ointment, cream oraerosol, using appropriate pharmaceutical carriers in accordance withthe administration method.

When the pharmaceutical composition is directly administered as aninjection to a patient, the polypeptide or its salt of the invention cancontinuously or intermittently administered in an amount of 10 to 5,000mg per kg of human body weight and per one day and by intravenous dripas a solution in physiological saline.

6. EXAMPLES

The present invention is further embodied in the examples shown belowwhich are not intended to limit the invention.

In the examples herein, the synthesis of polypeptide (1) is described.Additionally, the results of anti-HIV activity assays for thepolypeptides of the invention and known high endotoxin affinitypolypeptides are disclosed. Polypeptides of the invention have asignificantly higher anti-HIV activity than known high endotoxinaffinity polypeptides.

Apparatuses and reagents used in the following examples are as follows:

HPLC apparatus: Shimadzu Corporation, Model LC-6AD

Column of the apparatus: Asahipak ODP-90 (Asahi Chemical Industry Co.,Ltd.)

Fmoc amino acid and amino resin: produced by Watanabe ChemicalIndustries, Ltd.

Condensing agent: produced by Peptide Institute, INC. and AppliedBiosystems Japan

FAB-MS (FAB-mass spectrograph): VC Co. (USA), Model ZAB-SE

6.1. EXAMPLE 1 SYNTHESIS OF THE POLYPEPTIDE (1)

The synthesis of a polypeptide (1) which has the formula shown below isdescribed in Sections 6.1.1-6.1.7, infra. Polypeptides (2-13,22,23) andprecursor peptides of polypeptides(14-21,24, 25)(see Table I, supra forstructures) are synthesized using similar procedures. ##STR8##

In the above formula (1), Arg, Trp, Cys, Tyr, Lys, DLys and Pro denotethe aforementioned amino acid residue, and the solid line between theCys at the 3- and 11- positions denotes disulfide linkage.

6.1.1. INTRODUCTION INTO AN AMINOMETHYL RESIN OF Fmoc-DMBHA-CH₂ CH₂ COOH(3-(α-Fmoc-amino-4-methoxybenzyl)-4-methoxyphenyl)propionic acid!

270 mg (0.2 mmole) of an aminomethyl resin (0.74 meq/g) and 268.5 mg(0.5 mmole, 2.5 eq) of Fmoc-DMBHA-CH₂ CH₂ COOH (MW 537) were placed in asolid phase synthesizing column, and the condensation reaction wascarried out for 2 hours by the method of DIPCDI-HOBt in DMF according tothe method of Guo, L. et al. Chem. Pharm. Bull., 36, 4989 (1988)!.

After the completion of the condensation reaction, coupling was carriedout for the protection of the free amino groups using acetic anhydride(DMBHA resin).

6.1.2. INTRODUCTION OF ARGININE AT THE 12-POSITION INTO THE DMBHA RESIN

After removing the Fmoc groups from the DMBHA resin prepared in Section6.1.1., supra, with 20% piperidine/DMF, 2.5 equivalents (eq) ofFmoc-Arg(Pmc)-OH based on the DMBHA resin was added, and thecondensation reaction was carried out in DMF according to theDIPCDI-HOBt method.

The degree of progress of the condensation reaction was pursued bymeasurement according to the ninhydrin test of Kaiser, E. et. al. Anal.Biochem., 34, 595 (1970)!.

6.1.3. INTRODUCTION OF CYSTEINE AT THE 11-POSITION

After the removal of the Fmoc groups from the DMBHA resin prepared in6.1.2 with 20% piperidine/DMF, 2.5 eq of Fmoc-Cys (Trt)-OH based on theDMBHA resin was added, and condensation reaction was carried out in DMFby the method of DIPCDI-HOBt. The degree of progress of the condensationreaction was pursued similarly to 6.1.2., supra by measurement accordingto the ninhydrin test.

6.1.4. INTRODUCTION OF AMINO ACIDS FROM THE 10-TO 1-POSITIONS

Likewise as above, Lys(Boc), Arg(Pmc), Tyr(tBu), Pro, DLys(Boc),Lys(Boc), Arg(Pmc), Tyr(tBu), Cys(Trt), Trp, Arg(Pmc) and Arg(Pmc) weresuccessively introduced into the DMBHA resin to obtain a protectinggroup-protected peptide (1) resin.

Each amino acid condensation reaction in the solid phase synthesis wascarried out according to the operation conditions of the Table 2.

                  TABLE 2                                                         ______________________________________                                                                          Time ×                                                                  Repeat                                      Operation Reagent         Solvent number                                      ______________________________________                                        Removal of                                                                              20% piperidine/DMF                                                                            DMF     5 min. × 3                            Fmoc Group                                                                    Washing      --           DMF     1 min. × 6                            Condensation                                                                            Fmoc amino acid (2.5 eq)                                                                      DMF     .sup.   2 hr. × 1                     reaction  + DIPCDI + HOBt                                                     Washing      --           DMF     1 min. × 4                            ______________________________________                                    

6.1.5. PREPARATION OF THE POLYPEPTIDE (1) BY THE REMOVAL OF THEPROTECTING GROUPS. REMOVAL OF POLYPEPTIDE (1) FROM THE RESIN AND PARTIALPURIFICATION

The protecting group-protected polypeptide (1) resin was subjected to20% piperidine/DMF treatment to remove the Fmoc group, and thensubjected to reaction at 25° C. for 2 hours in a 1MTMSOTf-thioanisole/TFA(trifluoroacetic acid) system (10 ml oftrifluoroacetic acid in the presence of m-cresol (100 eq) andethanedithiol (300 eq)) per 100 mg of the resin. The resin was filteredoff from the reaction mixture and washed twice with 1 ml oftrifluoroacetic acid. 100 ml of ice-cooled dry ether was subsequentlyadded to mixture of the filtrate and the washing. The formed precipitatewas centrifuged, and the residue was separated from the supernatant bydecantation. The resulting residue was washed with cold ether, dissolvedin 10 ml of 4N AcOH and 830 mg, 80 eq of dithiothreitol was added. Themixture was stirred at room temperature overnight.

The reaction solution was centrifuged, the supernatant was treated withSephadex G-10 (3.7×5 cm), gel filtered with 4N acetic acid (AcOH), andthe fraction which flowed through the Sephadex was collected as the maineluate part and lyophilized to obtain as powder, a partially purifiednoncyclized polypeptide (1).

6.1.6. PREPARATION OF THE POLYPEPTIDE (1) BY AIR OXIDATION

A half amount of the flow-through fraction by the Sephadex gelfiltration was adjusted to pH 7.5 with concentrated aqueous ammonia, andsubjected to air oxidation by aeration to carry out the cyclizationreaction. After the completion of air oxidation, the cyclizedpolypeptide (1) was adsorbed onto 10 g of Diaion HP-20 resin, andsubsequently eluted with 60% CH₃ CN (in 1N AcOH).

The eluate was concentrated at room temperature under reduced pressureto remove CH₃ CN and then lyophilized to give powder. The powder wasdissolved in a small amount of water, and the solution was poured on anAsahipak ODP-90 column and purified by high performance liquidchromatography (HPLC-Model LC-6AD produced by Shimadzu Corp.) usinggradient elution with CH₃ CN to obtain the polypeptide (1) of a singlepeak in a yield of 27% (a value calculated based on the protectinggroup-protected polypeptide (1) resin).

6.1.7. ANALYSIS OF THE POLYPEPTIDE

The amino acid composition after acid hydrolysis according to the methodof Liu et al. J. Biol. Chem., 251, 1936 (1976)! and that obtained byleucine aminopeptidase digestion of the polypeptide purified as inSection 6.1.6., supra, were found to be well accorded within thecalculated value of the composition based on the amino acid sequence ofthe formula (1).

Further, the molecular weight value obtained by FAB-MS was 1996.3, whilethe calculated value of M+H!⁺ is 1996.1.

The specific rotation α!²⁰ _(D) of the obtained polypeptide was-17.2°(C=0.11, 1N acetic acid).

6.2 EXAMPLE 3 SYNTHESIS OF A POLYPEPTIDE (14) N-α-ACETYLATION OF THEAMINO TERMINAL AMINO ACID RESIDUE OF THE POLYPEPTIDE (1)!

The synthesis of a polypeptide (14) of the following formula 9 isdescribed in Sections 6.2.1-6.2.2: ##STR9##

In the above formula (14), Ac-Arg, Arg, Trp, Cys, Tyr, Lys, DLys and Prodenote the aforementioned amino acid residues, and the solid linebetween the Cys at 3- and 11- positions denote a disulfide bond.

6.2.1 ACETYLATION OF THE PARTIALLY PROTECTING GROUP-PROTECTED PEPTIDE(1) RESIN

1.301 g (0.25 mmol) of the protecting group-protected peptide (1) resinobtained at the step (4) (Section 6.1.4) of the aforementioned EXAMPLE 1was taken in a reaction vessel of a manual solid phase synthesis. Afterremoval of the Fmoc group, the N-terminal amino group was acetylatedaccording to the Hudson method J. Org. Chem., 53, 617, (1988)! to obtain1.241 g of an N-terminal α-amino acid-acetylated protectinggroup-protected peptide (1) resin (yield of dry weight, 100%). Theprocedure is summarized in the following Table 3.

                                      TABLE 3                                     __________________________________________________________________________                                          Time ×                            # Operation                                                                         Reagent              Solvent (volume)                                                                         Repeat number                           __________________________________________________________________________    1 Washing                  DMF (15 ml)                                                                              each 2 min. × 3                                              Isopropanol (15 ml)                                                           Dichloromethane (15 ml)                                                       DMF (15 ml)                                         2 Acylation                                                                         (i)                                                                              AcOH, 0.057 ml (1.0 mmol) +                                                                    DMF (6 ml) 3 min. × 1                              reaction           HOBt, 153 mg (1.0 mmol)                                    (ii)                                                                             BOP reagent*.sup.1 442.3 mg (1.0 mmol)                                                          DMF (2 ml) 3 min. × 1                              (iii)                                                                            Diisopropylethylamine                                                                           DMF (2 ml) 60 min. × 1                                0.35 ml (2 mmol)                                                     3 Washing                  DMF (15 ml)                                                                              each 2 min. × 3                                              Isopropanol (15 ml)                                                           Dichloromethane (15 ml)                                                       DMF (15 ml)                                        __________________________________________________________________________     *.sup.1 BOP reagent:                                                          Benzotriazole1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate

The acetylation was carried out by repeating the above operations 1 to3, until the ninhydrin reaction became no more positive.

6.2.2 PREPARATION OF THE PEPTIDE (14) BY THE REMOVAL OF THE PROTECTINGGROUPS AND THE RESIN OF THE N-TERMINAL AMINO ACID-ACETYLATED PROTECTINGGROUP-PROTECTED PEPTIDE (1) RESIN, PARTIAL PURIFICATION AND OXIDATION

The polypeptide (14) was prepared by the same way as described in 6.1.5and 6.1.6 supra. The specific rotation α!²⁰ _(D) of the polypeptide (14)was -18.3° (c=0.08, 1N acetic acid). The polypeptide (14) wasacid-hydrolyzed in 4M methanesulfonic acid containing 0.2% tryptamine at115° C. for 24 hours according to the method of Liu et al. J. Biol.Chem., 251, 1936 (1976)!. The result of the amino acid analysis accordedwell with the calculated value.

6.3 EXAMPLE 4 SYNTHESIS OF A POLYPEPTIDE (19)N-α-FLUORESCEINTHIOCARBAMOYL POLYPEPTIDE (1)!

The synthesis of a polypeptide (19) of the following formula 10 wascarried out by N-α-fluoresceinthiocarbamoylation of the amino terminalamino acid residue of the polypeptide (1). ##STR10## In the aboveformula (19), FTC-Arg, Arg, Trp, Cys, Tyr, Lys, DLys and Pro denote theaforementioned amino acid residues, and the solid line between the Cysat 3- and 11- positions denote a disulfide bond.

10 mg (3.9 μmol) of the acetic acid salt of the polypeptide (1) obtainedin 6.1.6 was dissolved in 1 ml of PBS buffer (phosphate buffered saline,pH 7.5). To the solution, 2.8 mg (7.2 μmol) of fluoresceinisothiocyanate (FITC) isomer-I (Wako Pure Chemical Ind., Ltd.) dissolvedin 1 ml of DMSO was added under ice-cooling. After 6 to 7 hours ofstirring at room temperature, the free amino group wasfluoresceinthiocarbamoylated.

The reaction mixture was applied to a Sephadex G-25(fine) columnequilibrated with 50 mM PB (phosphate buffer), pH 4.2!, preliminarilydesalted and fractionated. The peptide fraction was adsorbed on aSep-Pak C 18 plus ENV cartridge column (Millipore Co.), eluted with an80% acetonitrile/aqueous acetic acid solution pH 4.2 and freeze-dried toobtain 3.93mg of an acetic acid salt of the polypeptide (19) (yield,35%).

The specific rotation α!²⁰ _(D) of the acetic acid salt of the obtainedpolypeptide (19) was -5.9° (C=0.06, H₂ O).

The amino acid analysis of the acid hydrolysates of this compoundaccording to the Liu method showed one less observed value of arginineresidues than the calculated value for the polypeptide (1).

By thin layer chromatography (n-butanol:acetic acid: water =4:1:1) ofpartial acid hydrolysates of this compound in trifluoroacetic acid (roomtemperature for 2 hours), one main spot was detected, which correspondsto that of FTH-Arg (fluorescein arginine thiohydantoin).

The results of these analyses indicate that the α-amino group of the Nterminal arginine residue is selectively fluorescein thiocarbamoylated.

6.4 EXAMPLE 2 AND COMPARATIVE EXAMPLES 1 AND 2 ANTIVIRAL ACTIVITYAGAINST HUMAN IMMUNODEFICIENCY VIRUS (HIV)

The antiviral activity against HIV of the polypeptide (1) synthesized inExample 1 was tested and evaluated according to the following method.

HIV-infected MT-4 cells (2.5×104 cells/well, multiplicity of infection(MOI): 0.001) immediately after infection were added together with thetest substance with various changes of the concentration to a 96-wellmicrotitre plate. After incubation at 37° C. for 5 days in a CO₂incubator, the number of survivor cells was measured by the MTT methodPauwels et al.; J. Virol. Methods, 20, 309-321 (1988)!. The antiviralactivity is expressed as a concentration at which cell death due to HIVinfection is 50% inhibited (EC₅₀ : 50% effective concentration). On theother hand, in order to know the cytotoxicity of the test substance onthe MT-4 cells, virus-non-infected cells were incubated, likewise asabove, together with the test compound with various changes of theconcentration. The cytotoxicity is expressed as 50% cytotoxicconcentration (CC₅₀) due to the test substance. Further, the rough ratioof CC₅₀ to EC₅₀, (CC₅₀ /EC₅₀) is expressed as an effective ratio (SI).

The following formula 8 represents the peptide antiviral agent used incomparison with the polypeptide (1): Formula 8

    (Arg-Arg-Trp-Cys-Tyr-Arg-Lys-Cys-Tyr-Lys-Gly-Tyr-Cys-Tyr-Arg-Lys-Cys-Arg-NH.sub.2                                                     (T- 22)

Table 4 shows the EC₅₀, CC₅₀, and SI values of polypeptide (1), theabove peptide(T-22) and anti-HIV agent AZT.

                  TABLE 4                                                         ______________________________________                                                           CC.sub.50                                                                              EC.sub.50                                         Test compound      (μg/ml)                                                                             (μg/ml)                                                                            SI                                        ______________________________________                                        Example 2                                                                              Polypeptide(1)                                                                              49.8     0.0034                                                                              14,647                                  Comparative                                                                            T-22          54.1     0.0099                                                                               5,465                                  Example 1                                                                     Comparative                                                                            AZT           6.68     0.0001                                                                              66,800                                  Example 2                                                                              (azidothymidine) μM                                               ______________________________________                                    

The above table apparently shows that the polypeptide (1) of theinvention has the same cytotoxicity as that of T-22, whose anti-HIVactivity was previously revealed, but exhibits an antiviral activity atone-third the concentration of T-22. Even though 4 amino acid residueshave been removed from the peptide and the molecular weight has beenlowered, the polypeptide of the invention exhibited an even higheractivity.

In comparison with the azidothymidine(AZT), the EC₅₀ value of thepolypeptide (1) is slightly higher, but it exhibited extremely lowcytotoxicity. Therefore we expect that it can be used as a saferanti-HIV agent.

6.5 EXAMPLE 5 THE CHARACTERISTICS OF THE POLYPEPTIDES AND THEIR ANTI-HIVACTIVITY

Table 5 shows the structural formula and the characteristics of thepolypeptides of the invention prepared by the procedure of Examples 1, 3and 4. Table 5 also shows anti-HIV activity of the polypeptides testedand evaluated according to the method of Example 2.

                                      TABLE 5                                     __________________________________________________________________________                                                Physical Property                 Compound (Mame) & Structure                  α!.sub.D.sup.20-22                                                             (Conc.,                   __________________________________________________________________________                                                        Solvent)                   (1) Arg--Arg--Trp--Cys--Tyr--Arg--Lys--DLys--Pro--Tyr--Arg--Lys--Cys--Arg    --NH.sub.2                                  -17.2   (0.11, 1NAcOH)             (3) Arg--Arg--Trp--Cys--Tyr--Arg--Lys--Lys--Gly--Tyr--Arg--Lys--Cys--Arg-    -NH.sub.2                                   -0.9    (0.04, 1NAcOH)             (5) Arg--Arg--Trp--Cys--Tyr--Arg--Lys--Pro--DLys--Tyr--Arg--Lys--Cys--Arg    --NH.sub.2                                  -12.1   (0.06, 1NAcOH)             (6) Arg--Arg--Trp--Cys--Tyr--Arg--Lys--DOrn--Pro--Tyr--Arg--Lys--Cys--Arg    --NH.sub.2                                  -12.5   (0.10, 1NAcOH)            (14) Ac--Arg--Arg--Trp--Cys--Tyr--Arg--Lys--DLys--Pro--Tyr--Arg--Lys--Cys-    -Arg--NH.sub.2                              -18.3   (0.08, 1NAcOH)            (17) Myr--Arg--Arg--Trp--Cys--Tyr--Arg--Lys--DLys--Pro--Tyr--Arg--Lys--Cys    --Arg--NH.sub.2                             -19.1   (0.08, 1NAcOH)            (19) FTC--Arg--Arg--Trp--Cys--Tyr--Arg--Lys--DLys--Pro--Tyr--Arg--Lys--Cys    --Arg--NH.sub.2                             -5.9    (0.06, H2O)                ##STR11##                                  -13.6   (0.07, 1NAcOH)             ##STR12##                                  -12.7   (0.10,                    __________________________________________________________________________                                                        1NAcOH)                                                               Anti-HIV Activity                 Compound (Mame) & Structure                 CC.sub.50 (μg/ml)                                                                EC.sub.50                                                                           SI (CC.sub.50                                                                 /EC.sub.50)           __________________________________________________________________________     (1) Arg--Arg--Trp--Cys--Tyr--Arg--Lys--DLys--Pro--Tyr--Arg--Lys--Cys--Arg    --NH.sub.2                                  65.37 0.0195                                                                              3352                   (3) Arg--Arg--Trp--Cys--Tyr--Arg--Lys--Lys--Gly--Tyr--Arg--Lys--Cys--Arg-    -NH.sub.2                                   198.18                                                                              0.057 3477                   (5) Arg--Arg--Trp--Cys--Tyr--Arg--Lys--Pro--DLys--Tyr--Arg--Lys--Cys--Arg    --NH.sub.2                                  101.20                                                                              0.076 1321                   (6) Arg--Arg--Trp--Cys--Tyr--Arg--Lys--DOrn--Pro--Tyr--Arg--Lys--Cys--Arg    --NH.sub.2                                  55.93 0.021 2728                  (14) Ac--Arg--Arg--Trp--Cys--Tyr--Arg--Lys--DLys--Pro--Tyr--Arg--Lys--Cys-    -Arg--NH.sub.2                              78.14 0.024 3256                  (17) Myr--Arg--Arg--Trp--Cys--Tyr--Arg--Lys--DLys--Pro--Tyr--Arg--Lys--Cys    --Arg--NH.sub.2                             106.60                                                                              0.055 1838                  (19) FTC--Arg--Arg--Trp--Cys--Tyr--Arg--Lys--DLys--Pro--Tyr--Arg--Lys--Cys    --Arg--NH.sub.2                             38.47 0.001 39285                  ##STR13##                                  44.26 0.030 1475                   ##STR14##                                  138.07                                                                              0.0165                                                                              11095                 Comparative Example:                                                          T-22                                        49.4  0.017 2889                  AZT                                         8.53  0.0014                                                                              6093                  __________________________________________________________________________

Unless otherwise specifies, the Cys at the 3- and 11- positions of theexample compounds presented in the above tables are linked by adisulfide bond. Further, in the above table, "AZT" denotesazidothymidine (commonly known as zidovudine), and T-22 denotes thepolypeptide represented in formula 8.

Effectiveness of the invention!

The present invention provides a novel polypeptide having antiviralactivity against human immunodeficiency virus (HIV) and it has makepossible to give hydrophylicity, affinity for lipids, more higheractivity and useful for elucidating the manifestation mechanism ofanti-HIV activity.

We claim:
 1. A polypeptide represented by the following formula##STR15## or salt thereof in which A₁ is a basic amino acid residue, ora peptide residue having at least two basic amino acids, selected fromthe group consisting of lysine, arginine and ornithine, said basic aminoacid residue or peptide residue in which N-α hydrogen atom of aminoterminal end of said amino acid residue may be replaced with an acylgroup or a substituted thiocarbamoyl group, forming N-α acyl substitutedbasic amino acid residue, N-α acyl substituted peptide residue, N-αsubstituted thiocarbamoyl group substituted basic amino acid residue orN-α substituted thiocarbamoyl group substituted peptide residue;A₂ is atyrosine or phenylalanine residue; A₃ is a lysine or arginine residue;A₄ is an --OH (derived from a carboxyl group) or an --NH₂ (derived froman acid amide group); X is a peptide residue selected from the groupconsisting of the peptides represented by D-ornithyl-proline,prolyl-D-ornithine, D-lysyl-proline, prolyl-D-lysine, D-arginyl-proline,prolyl-D-arginine, glycyl-ornithine, ornithyl-glycine, glycyl-lysine,lysyl-glycine, glycyl-arginine and arginyl-glycine, in which thehydrogen atom of the ω-amino group of D-lysine, L-lysine, D-ornithineand L-ornithine may be replaced by an ω-amino acyl group, and saidpeptide residue is connected to the amino acid residues at the 6th andthe 8th positions via peptide bond per se; Trp is a tryptophan residue;and Cys is a cysteine residue.
 2. The polypeptide or salt thereof ofclaim 1 in which A₁ is at least one basic amino acid selected from thegroup consisting of lysine, arginine and ornithine.
 3. The polypeptideor salt thereof of claim 1 in which A₁ is peptide residue of two basicamino acids, in which said amino acids are selected from the groupconsisting of lysine, arginine and ornithine.
 4. The polypeptide or saltthereof of claim 1 in which A₁ is at least one basic amino acid residuein which N-α hydrogen atom of the amino terminal end of the amino acidresidue is replaced with an acyl group or a substituted thiocarbamoylgroup.
 5. A polypeptide represented by the following formula ##STR16##or salt thereof in which A₁ is a basic amino acid residue, or a peptideresidue having at least two basic amino acids, selected from the groupconsisting of lysine, arginine and ornithine, said basic amino acidresidue or peptide residue in which N-α hydrogen atom of amino terminalend of said amino acid residue may be replaced with an acyl group or asubstituted thiocarbamoyl group, forming N-α acyl substituted basicamino acid residue, N-α acyl substituted peptide residue, N-αsubstituted thiocarbamoyl group substituted basic amino acid residue orN-α substituted thiocarbamoyl group substituted peptide residue;A₂ is atyrosine or phenylalanine residue; A₃ is a lysine or arginine residue;A₄ is an --OH (derived from a carboxyl group) or an --NH₂ (derived froman acid amide group); X is a peptide residue selected from the groupconsisting of the peptides represented by D-ornithyl-proline,prolyl-D-ornithine, D-lysyl-proline, prolyl-D-lysine, D-arginyl-proline,prolyl-D-arginine, glycyl-ornithine, ornithyl-glycine, glycyl-lysine,lysyl-glycine, glycyl-arginine and arginyl-glycine, in which thehydrogen atom of the ω-amino group of D-lysine, L-lysine, D-ornithineand L-ornithine may be replaced by an ω-amino acyl group, and saidpeptide residue is connected to the amino acid residues at the 6th andthe 8th positions via peptide bond per se; Trp is a tryptophan residue;and Cys is a cysteine residue; and the cysteine residues at the 3- and11- positions are linked through a disulfide linkage.
 6. Apharmaceutical composition for inhibiting HIV activity in a patientcomprising an effective amount of the polypeptide or salt thereof ofclaim 1 and a pharmaceutical carrier.
 7. A pharmaceutical compositionfor inhibiting HIV activity in a patient comprising an effective amountof the polypeptide or salt thereof of claim 5 and a pharmaceuticalcarrier.